The research program will produce and employ immunochemical reagents to study the EGF receptor in cultured cells. Using highly purified, by affinity chromatography, preparations of the EGF receptor from A-431 tumor cells, polyclonal antibodies will be prepared in rabbits and monoclonal antibodies will be produced by hybridoma technology. The polyclonal antisera will be characterized and employed to study the metabolism of the EGF receptor during the "normal" growth of culture cells and subsequently during alterations of cell culture conditions which are known to affect receptor activity - "down regulation" by EGF and possibly other polypeptide hormones, sarcoma virus transformation, modulation of protein synthesis, treatment with vitamin A. To measure receptor synthesis, degradation or recycling under these conditions, cell protein will be labeled with radioactive amino acids and immunoprecipitation with antibodies employed to detect incorporation or turnover of radioactivity in the EGF receptor. These studies will provide information concerning EGF receptor metabolism in a definitive manner not dependent on binding activity or the use of inhibitors. Specific antibodies also will be utilized to morphologically localize EGF receptors, by electron microscopic methods, in intracellular compartments. The monoclonal antibodies will be developed in the hope of obtaining a completely specific antibody for the EGF receptor and of obtaining a "library" of antibodies which mimic or inhibit the various mitogenic activities of EGF in vivo and in vitro. Monoclonal antibodies will be employed in a study of match tryptic fragments of the EGF receptor-kinase with various monoclonal antibodies. This study may relate biological activities of the antibodies to structural segments of the receptor-kinase. Monoclonal antibodies also will be employed to address questions of receptor functional and chemical heterogeneity and will be used as reagents in the further purification of the EGF receptor and kinase.